Evaluation of Urease Activity Assessed on Urea Concentration Using Tannic Acid-Silver Nitrate Colorimetric Method

Urease, a nickel-dependent enzyme, hydrolyses urea into ammonia and carbon dioxide. The unhydrolyzed urea, in the presence of silver nitrate and tannic acid, forms a yellow-brown complex. This study examines the effect of urea concentration on urease activity using a colorimetric method that involves tannic acid and silver nitrate. The results revealed that low concentrations of urea, less than or equal to 1.00 mM, may limit substrate availability, leading to an underestimation of enzyme activity. An optimal urea concentration of 3.00 mM produced the highest absorbance, 0.949, indicating maximal urease activity (V_max). The high urea concentrations, greater than or equal to 4.00 mM, resulted in lower absorbance, possibly due to pH shifts or altered reaction kinetics. Low urea concentrations show narrow or sharp SPR peaks at around 414 nM, whereas higher concentrations display broader or wider peaks. The Lineweaver-Burk method shows that the enzyme kinetics of the urea hydrolysis efficiency were greatest at about 3 mM. At this concentration, catalytic efficiency and enzyme/substrate binding were very high (i.e., low Km and near-saturation V_max). Higher substrate concentrations led to decreased reaction rates. The changing wavelengths suggested substrate inhibition and possible sensor behavior related to nanoparticles. These results indicate that this approach is viable for a very sensitive urea biosensor.